Tolerizing agents

ABSTRACT

Described herein is the development of fusion proteins useful for inducing tolerance in a subject. In particular embodiments, the tolerizing agents are useful for influence autoimmune, inflammatory, and/or allergic reactions. Example tolerizing fusion proteins contain a targeting portion (which delivers the fusion protein) and a toleragen or allergen or other antigen to which tolerance is desired in a subject. In particular examples, it is demonstrated that a pσ1 fusion protein, when administered orally, facilitates systemic and mucosal tolerance. Also described is the nasal delivery of fusion proteins, for instance for restoring immunogenicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 12/294,380, filedSep. 24, 2008, which is the U.S. National Stage of InternationalApplication No. PCT/US2007/065278, filed Mar. 27, 2007, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. provisional application No. 60/786,446, filed Mar. 27,2006. All of the above-listed applications are hereby incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contractsAI018958, DE012242, AI043197, DC004976, and DE013812 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE DISCLOSURE

This disclosure relates to agents and compositions useful in stimulatingtolerance to an immunogen. In particular, it relates to mucosal targetedfusion proteins that can be applied, for instance, through oral and/ornasal routes to tolerize a subject.

BACKGROUND OF THE DISCLOSURE

Oral administration of a single high dose or repeated low doses ofprotein has been shown to induce systemic unresponsiveness, presumablyin the presence of mucosal IgA antibody responses (Challacombe et al.,J. Exp. Med. 152:1459-1472, 1980; Mestecky et al., “The mucosal immunesystem.” In Fundamental Immunology. Paul, ed. Lippincott Williams &Wilkins, Philadelphia, Pa., 965-1020, 2003). In earlier studies, thistype of immune response was dubbed oral tolerance and the concept wasused to refer specifically to immune responses elicited inmucosa-associated as opposed to systemic lymphoid tissues (Tomasi,Transplantation 29:353-356, 1980). However, previous studies showed thattolerance induction occurred in the mucosal effector lymphoid tissues(Kato et al., J. Immunol. 166:3114-3121, 2001). Thus, mice fed largeamounts of ovalbumin (OVA) prior to oral challenge with OVA plus nativecholera toxin (CT) as mucosal adjuvant exhibited antigen (Ag)-specificunresponsiveness in both systemic and mucosal compartments, while thosefed PBS showed high levels of secretory (S)-IgA Ab responses (Kato etal., J. Immunol. 166:3114-3121, 2001).

This unique response is an important natural physiological mechanismwhereby the host presumably avoids development of hypersensitivityreactions to many ingested food proteins and other antigens (Garside etal., Gut 44:137-142, 1999). Thus, tolerance (or systemicunresponsiveness) represents the most common response of the host to theenvironment. In addition to showing tolerance to several thousanddifferent food proteins, the host tolerates indigenous microflora whichcolonize the large intestine. Further, the development of mucosaltolerance against pollen and dust antigens could also be essential forthe inhibition of allergic reactions, including IgE-mediatedhypersensitivity. Indeed, tolerance is so strong that oral immunizationonly succeeds in inducing mucosal and systemic immunity when potentmucosal adjuvants, vectors or other special delivery systems areemployed (Fujihashi et al., Acta. Odontol. Scand. 59:301-308, 2001).

It is now generally agreed that oral tolerance is established andmaintained at the level of T cells (Holt, Allergy 53:16-19, 1998;MacDonald, Curr. Opin. Immunol. 10:620-627, 1998; Mayer, Clin. Immunol.94:1-8, 2000; Strobel & Mowat, Immunol. Today 19:173-181, 1998; Stroberet al., J. Clin. Immunol. 18:1-30, 1998; Wardrop & Whitacre, Inflamm.Res. 48:106-119, 1999; Weiner et al., Annu. Rev. Immunol. 12:809-837,1994). Recent studies have identified dendritic cells as key players inthe direct or indirect (via T cells) induction of oral tolerance (Mowat,Nat. Rev. Immunol. 3:331-341, 2003; Kato et al., Int. Immunol.15:145-158, 2003; Nagler-Anderson & Shi, Crit. Rev. Immunol. 21:121-131.2001; Viney et al., J. Immunol. 160:5815-5825, 1998; Williamson, J.Immunol. 163:3668-3675, 1999; Weiner, Immunol. Rev. 182:207-214, 2001).Though the precise mechanisms by which oral delivery of Ag elicits astate of systemic unresponsiveness are not fully understood, the dosageof Ag has been shown to be an important factor (Friedman & Weiner, Proc.Natl. Acad. Sci. (USA) 91:6688-6692, 1994). For example, a high oral Agdose leads to T cell clonal deletion or anergy, which is characterizedby inhibition of both Ab- and cell-mediated immune (CMI) responses(Melamed & Friedman, Eur. J. Immunol. 23:935-942, 1993; Whitacre et al.,J. Immunol. 147:2155-2163, 1991; Chen et al., Nature 376:177-180, 1995).On the other hand, repeated delivery of low doses of protein inducescytokine-mediated active immune suppression characterized by thepresence of regulatory T cells, which include TGF-β-producing Th3 cellsand IL-10-producing T regulatory one (Tr1) cells or CD4⁺ CD25⁺ Tregulatory (Treg) cells (Chen et al., Science 265:1237-1240, 1994; Grouxet al., Nature 389:737-742, 1997; Nagler-Anderson et al., Nat. Immunol.5:119-122, 2004). Regulatory-type T cells were first rediscovered asacquired-type Tr1 cells playing a central role in suppressinginflammatory bowel disease development (Groux et al., Nature389:737-742, 1997). Acquired-type Treg cells, which differentiate fromnaïve T cells, regulate tolerance to food Ags, bacterial flora andpathogens by producing suppressive cytokines such as TGF-β1 and IL-10(Cottrez & Groux, Transplantation 77:S12-15, 2004). In contrast,naturally occurring CD4⁺ CD25⁺ T cells or innate-type Treg cells, whichare also suppressive, control the proliferation, expansion anddifferentiation of naïve T cells in a direct cell contact manner(Dieckmann et al., J. Exp. Med. 196:247-253, 2002) and migratepreferentially to lymphoid tissues, mainly the spleen (Cottrez & Groux,Transplantation 77:S12-15, 2004).

In addition to CD4⁺ T cell function, gut-associated lymphoreticulartissues (GALT) play critical roles in the induction of oral tolerance.In this regard, our previous studies showed that Peyer's patch(PP)-deficient (PP-null) mice generated by in utero treatment of motherswith lymphotoxin beta-receptor (LTβR)-immunoglobulin (Ig) fusion proteinfailed to exhibit systemic unresponsiveness to oral protein antigens(Ag) such as OVA (Fujihashi et al., Proc. Natl. Acad. Sci. (USA)98:3310-3315, 2001). In contrast, others reported that PPs were notrequired for the induction of systemic tolerance (Spahn et al., Eur. J.Immunol. 32:1109-1113, 2002). Recent studies have shown the importanceof Ag-specific CD4⁺ CD25⁺ Treg cell clones from PPs in oral toleranceinduction. Thus, Treg cells from PP of mice given a high dose ofβ-lactoglobulin produced high levels of TGF-β1, and adoptive transfer ofthese clones reduced Ag-specific plasma IgG Ab responses (Tsuji et al.,Int. Immunol. 15:525-534, 2003). Despite these compelling studies, theprecise cellular and molecular mechanisms and the role of PPs in theinduction of systemic and mucosal unresponsiveness still remain to beelucidated.

Adenoviruses enter the host via attachment to the mucosal epithelia byits protein known as “fiber protein”. Likewise, reoviruses infect thehost by attaching to M cells via a protein called “protein al” (pσ1; Wuet al., Proc. Natl. Acad. Sci. (USA) 98:9318-9323, 2001; Rubas et al., JMicroencapsul 7:385-395, 1990). These attachment proteins of adenovirusssp. and reovirus ssp. are well known, and share a strikingly structuralsimilarity despite lack of homology at the primary structure level. Bothproteins are composed of a N-terminal shaft followed by a C-terminalglobular domain, sometimes referred to as “head” or “knob”. The shaftinserts into the viral capsids, while the globular domains contain thecell-specific targeting regions. For both of these viruses, the shaftcontains a domain that causes the protein to form homotrimers, theactive form of the protein.

Incorporation of pσ1 into liposomes allows the latter to bind to mouse Lcells and rat Peyer's patches (Rubas et al., J Microencapsul 7:385-395,1990), and the recombinant pσ1 is also known to bind to NALT M cells (Wuet al., Gene Ther. 7:61-69, 2000; Wu et al., Proc. Natl. Acad. Sci.(USA) 98:9318-9323, 2001). In marked contrast to results seen when DNAis given alone, immunization with DNA complexed topoly-L-lysine-conjugated pσ1 leads to elevated S-IgA and plasma IgG Abresponses (Wu et al., Proc. Natl. Acad. Sci. (USA) 98:9318-9323, 2001).

There exists a need to develop agents that can stimulate or causetolerance in a subject to an immunogen. It is to such agents, andcompositions comprising such, that this disclosure is drawn.

OVERVIEW OF REPRESENTATIVE EMBODIMENTS

Described herein is the development of M cell-targeting Ag deliverysystems using recombinant reovirus pσ1. Recombinant pσ1 of reovirus hasbeen genetically fused to OVA (OVA-pσ1). It is demonstrated that thisfusion protein, when administered orally, facilitates systemic andmucosal tolerance induction by innate- and/or acquired-types of Tregcells. Also described is the nasal delivery of a OVA-pσ1(m) orOVA-pσ1(Δ) fusion protein for restoring OVA immunogenicity.

Thus, there is provided herein a new approach to delivering highlyvirulent and antigen-specific tolerizing agents, which uses a ligand(such as a mucosal targeting ligand) fused to a specific antigen toinduce host unresponsiveness solely to that antigen. The ligand portionof the protein can be fused a broad range of antigens (toleragens),enabling the generation of tolerance to a number of autoimmune diseaseantigens, inflammatory disease antigens, allergens, and biologicaltherapeutic molecules (e.g., botulinum toxin), for instance. The fusionproteins are capable of regulating peripheral tolerance subsequent tonasal or oral application.

The tolerizing fusion proteins provided herein can be used in varioustolerance applications, including but not limited to treatment oramelioration of autoimmune diseases, inflammatory diseases, allergicreactions, graft or transplant rejection, and so forth. In addition, theprovided proteins and methods of their use permit continuous or on-goingtreatment of a subject with a biological therapeutic agent. For example,tolerance has been demonstrated in mice challenged with ovalbumin ormyelin proteins, the latter being useful for treatment against multiplesclerosis.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Optimization of OVA-pσ1 for the induction of mucosal tolerance(A). BALB/c mice were fed a single dose of OVA-pσ1 [1000 (□) 500 (

) or 100 (

) μg] prior to oral challenge with OVA (1 mg) plus CT as adjuvant (10μg) three times at weekly intervals. In some experiments, mice weregiven three separate doses of 100 μg of OVA-pσ1 (▪) at daily intervalsbefore oral challenge. Plasma and fecal extract samples were collectedseven days after the last oral challenge and subjected to OVA-specificELISA. As a control group, mice were fed PBS prior to oral challengewith OVA plus CT (dotted line). The results represent the meanvalues±SEM for 12 mice in each experimental group and were taken fromthree separate experiments.

FIG. 2. Numbers of OVA-specific AFCs in various lymphoid tissues. BALB/cmice were fed three separate doses of OVA-pσ1 (100 μg) at weeklyintervals prior to oral challenge with OVA (1 mg) plus CT (10 μg). Ascontrols, mice were fed PBS prior to oral challenge with OVA plus CT.Mononuclear cells from spleen, MLNs and iLP were isolated seven daysafter the last oral challenge and subjected to OVA-specific ELISPOTassays in order to detect anti-OVA IgM (□) IgG (

) and IgA (▪) AFCs. The results represent the mean±one standard error ofthe mean (SEM) for 12 mice in each experimental group and are taken fromthree separate experiments.

FIG. 3. OVA-specific DTH responses and OVA-specific CD4⁺ T cellproliferative responses. (A) Six days after the last oral challenge,both OVA-pσ1 (□)- and PBS (▪)-fed groups of mice were injected with 10μg of OVA in 20 μl of PBS into the right ear pinna. PBS (20 μl) wasadministered to the left ear pinna as a control. The thickness of theear was measured 24 hours later with an upright dial thickness gauge.The DTH response was expressed as the increase in ear swelling afterchallenge with Ag after subtraction of swelling in the control site. (B)Seven days after the last oral challenge, CD4⁺ T cells were purifiedfrom both OVA-pσ1 (□)- and PBS (▪)-fed mice. Purified CD4⁺ T cellfractions were cultured with or without one mg/ml of OVA in the presenceof APCs. An aliquot of 0.5 μCi of tritiated [³H]-thymidine was addedduring the final 18 hours of incubation, and the amount of[³H]-thymidine incorporation was determined by scintillation counting.The stimulation index was determined as cpm of wells with Ag/cpm ofwells without Ag (controls). The levels of [³H] TdR incorporated in eachcontrol well ranged from 500 to 1,000 cpm. The results represent themean values±1 SEM from three separate experiments (triplicatewells/experiment).

FIG. 4. Detection of frequency of OVA-specific CD4^(′) T cells.Mononuclear cells from the spleen, MLNs, PPs and iLPs of mice fedOVA-pσ1 or PBS were stained with FITC-conjugated anti-CD4 mAb andPE-labeled OVA/I-A^(d) tetramer. Samples were subjected to flowcytometric analysis using FACSCALIBUR™. The results represent typicalresults and are taken from one of three separate experiments.

FIG. 5. TGF-β1 and IL-10 production by CD4⁺ CD25⁺ T cells. Mice were fed100 μg of OVA-pσ1 (□) or PBS (▪) before being orally immunized weeklyfor three weeks with 1 mg of OVA plus 10 μg of CT. (A) CD4⁺ CD25⁺ Tcells were purified from PPs, MLNs and spleen by flow cytometry andcultured with 1 mg/ml of OVA in the presence of irradiated APCs. Thelevels of TGF-β1 in the culture supernatants were determined by aTGF-β1-specific ELISA. (B) Interleukin-10 production by CD4⁺ CD25⁺ Tcell subsets in MLNs and spleen were determined by intercellularanalysis. Mononuclear cells were incubated with ionomycin (1 μg/ml,SIGMA, St. Louis, Mo.) and phorbol 12-myristate 13-acetate (PMA, 25ng/ml, SIGMA) for 6 hours and then stained with PE-labeled anti-CD4,biotinylated anti-CD25 mAbs followed by Cy5.5-streptavidin. Thesesamples were further stained intracellularly with ALEXA FLUOR®488-labeled anti-IL-10 mAb (JES5-16E3). The results represent the meanvalues ±1 SEM from three separate experiments.

FIG. 6. Protein σ1 (pσ1) variants described here: recombinant pσ1;pσ1(m) has a mutagenized sialic acid binding domain (SABD); OVA-pσ1;OVA-pσ1(m) has a mutagenized SABD; and OVA-pσ1(Δ) lacks its shaft andSABD.

FIG. 7. OVA-pσ1 fails to elicit delayed-type hypersensitivity (DTH)responses to OVA. Mice were given three i.n. doses of OVA-pσ1+CT,OVA-pσ1(Δ)+CT, OVA+CT, or OVA alone on days 0, 7, and 14. On day 42,mice were challenged with 10 μg of OVA into one ear pinna and withsterile PBS in the other, and differences in ear swelling were measured24 hours later. Compared to mice dosed with OVA+CT: *P<0.001,***P=0.012, and NS=not significant. Mice i.n. dosed with OVA only wasnot significantly different from mice i.n. dosed with OVA-pσ1+CT; micei.n. dosed with OVA-pσ1(Δ)+CT were significantly different fromOVA-pσ1+CT-dosed mice (**P=0.002). Depicted were the means±SEM ofindividual mice from two experiments.

FIG. 8. CD4⁺ T cells from mice nasally dosed with OVA-pσ1 mediate OVAunresponsiveness following adoptive transfer and peripheral OVAchallenge. DO11.10 TCR CD4⁺ T cells were adoptively transferred intonaive BALB/c mice, and subsequently dosed i.n. with PBS, 400 μg OVA, or80 μg OVA-pσ1 or i.m. with 400 μg OVA. Three days later, CLN CD4⁺ Tcells were adoptively transferred into naive BALB/c mice, and 24 hourslater, they were challenged with 100 μg in incomplete Freund's adjuvant.CD4⁺ T cells were isolated from the CLN and spleen five days later, andthen cultured with mitomycin C-treated feeder (T cell-depleted) cellswithout or with 1.0 mg OVA for five days. ³H-thymidine incorporation wasmeasured and expressed as a stimulation index (SI). For CLN, ^(§)P≦0.001vs. i.m. OVA; for spleen, **P=0.003, ***P=0.006 vs. i.m. OVA.

FIG. 9. Modification of OVA-pσ1 with encephalitogenic peptides retainsability to induce unresponsiveness to OVA. (FIG. 9A) OVA-pσ1 wasgenetically modified at its N-terminus to express 2 copies of theencephalitogenic peptide derived from proteolipid protein (PLP)₁₃₉₋₁₅₁separated by an irrelevant peptide sequence ((MOG)₃₅₋₅₅); this fusionprotein is referred to as AR1. (FIG. 9B-E) C57BL/6 mice were nasallydosed on days 0, 7, & 14 with 100 μg of AR1, and (FIG. 9B) plasma IgGand (FIG. 9C) IgA and (FIG. 9D) copro-IgA were measured by OVA-specificELISA. Only the OVA+CT group showed anti-OVA Abs. *P<0.001. On days 21and 27, mice were challenged i.n. with OVA+CT. Then on day 35, DTH testwas performed as described in FIG. 3 (10 μg of OVA was injected into theleft ear pinna and PBS alone into the right ear pinna as a control. Earswelling was measured 24 and 48 hrs later, and differences recorded.).Again, only the OVA+CT group showed a DTH response upon OVA challenge.Thus, these data show that the genetic fusion of the described peptidesdid not interfere with the OVA-pσ1 core.

FIG. 10. Mice nasally dosed with AR1 (a tolerogenic vaccine for EAE) areprotected against EAE challenge. (FIG. 10A) SJL/J mice were dosed withproteolipid protein peptide (PLP₁₃₉₋₁₅₁)₂:OVA-pσ1 (AR1; n=8), asdescribed in FIG. 9, and were challenged s.c. with PLP₁₃₉₋₁₅₁ inmodified complete Freund's adjuvant+i.p. pertussis toxin (PT). A seconddose of PT was given i.p. two days later and mice were followed fordisease. As a positive oral tolerance control (n=5), one group of micewas orally tolerized with myelin basic protein (MBP) since these micewere protected (p<0.001) as were mice dosed with AR1 (p<0.001) whencompared to PBS-dosed (diseased) mice (n=8). (FIG. 10B) C57BL/6 micewere nasally dosed with 50 μg myelin oligodendrocyte glycoprotein₂₉₋₁₄₆genetically fused to pσ1 (MOG-pσ1) or to OVA-pσ1 (MOG:OVA-pσ1) threetimes at weekly intervals, and then 1 wk after the last i.n. dose, micewere challenged s.c. with 150 μg MOG₃₅₋₃₃ on day 0 and 7 of challenge,and given i.v. PT on days 0 and 2. Both the MOG-pσ1 (n=5) andMOG:OVA-pσ1 (n=5) protected mice (p<0.001) when compared to PBS-dosedmice (n=5).

FIG. 11. Protection against PLP₁₃₉₋₁₅₁ challenge is attributed to thestimulation of the regulatory cytokines, IL-4, IL-10, and TGF-β. SJLmice were dosed with AR1, OVA-pσ1, or PBS as described in FIG. 9. Micewere then challenged with PLP₁₃₉₋₁₅₁ peptide as described in FIG. 10.HNLN, spleens, and MLN were harvested at peak of disease (day 14) andpurified CD4⁺ T cells were restimulated with PLP₁₃₉₋₁₅₁ peptide for 2days, and evaluated in a cytokine ELISPOT. PBS- and OVA-pσ1-dosed(unprotected mice) showed elevated (FIG. 11A) IFN-γ and (FIG. 11B) IL-17cytokine-forming cells (CFC), and no (FIG. 11C) IL-4, (FIG. 11D) IL-10,or (FIG. 11E) TGF-β CFC. In contrast, AR1-dosed (tolerized) mice showedelevated IL-4, IL-10, and TGF-β CFC and no IFN-γ or IL-17 CFC. Thus,only AR1 mice were protected against challenge, and tolerance induced toirrelevant protein (OVA-pσ1) did confer protection. *P<0.001 betweenAR1-dosed mice versus PBS-dosed mice.

FIG. 12. Single nasal or oral dose with MOG-pσ1 protects C57BL/6 miceagainst challenge with MOG₃₅₋₅₅. Mice (5/group) were dosed once (FIG.12A) nasally or (FIG. 12B) orally with 10, 50, or 100 μg of _(MOG)₂₉₋₁₄₆-pσ1 (MOG-pσ1) or with PBS, and 10 days later challenged withMOG₃₅₋₅₅ per description for FIG. 10. In a dose-dependent fashion,protection against autoimmune challenge showed protection, but the 50 μgdose conferred the best protection with no disease, while minimaldisease was observed at the 10 or 100 μg doses. Thus, pσ1 delivery is aneffective means to deliver auto-antigens to the mucosa for thedevelopment of tolerance to self antigens.

FIG. 13. Nasal treatment of C57BL/6 mice with MOG-pσ1 results indiminished EAR Groups of C57BL/6 mice were induced with EAE as describedin FIG. 10B using MOG₃₅₋₅₅ peptide. On day 7, one group of mice werenasally dosed with 50 μg MOG-pσ1 or PBS, and disease course followed. Ondays 10 and 17, separate groups of mice were nasally dosed with 50 μgMOG-pσ1, and disease course followed. Mice treated with MOG-pσ1 showedeither no EAE or only minor disease in some mice. Thus, MOG-pσ1 can beused therapeutically to treat EAE.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three-letter code for amino acids, as defined in 37 C.F.R.§1.822. Only one strand of each nucleic acid sequence is shown, but asappropriate in context the complementary strand is understood asincluded by any reference to the displayed strand. The Sequence Listingis submitted as an ASCII text file, created on February, 7, 2011, 58.4KB, which is incorporated by reference herein. In the accompanyingsequence listing:

SEQ ID NO: 1 shows the nucleic acid sequence encoding adenovirus 2 fiberprotein (HAD278923).

SEQ ID NO: 2 shows the protein sequence of adenovirus 2 fiber protein.

SEQ ID NO: 3 shows the nucleic acid sequence encoding reovirus type 3sigma 1 (haemagglutinin) (RET3S1).

SEQ ID NO: 4 shows the amino acid sequence of reovirus type 3 sigma 1(haemagglutinin).

SEQ ID NO: 5 shows the nucleic acid sequence encoding adenovirus 16fiber protein (AX034843).

SEQ ID NO: 6 shows the amino acid sequence of adenovirus 16 fiberprotein.

SEQ ID NO: 7 shows the nucleic acid sequence encoding adenovirus 35fiber (fiber) protein (30827 to 31798 of BK005236).

SEQ ID NO: 8 shows the amino acid sequence of adenovirus 35 fiberprotein (30827 to 31798 of BK005236).

SEQ ID NO: 9 shows the nucleic acid sequence encoding adenovirus 37fiber protein (x94484).

SEQ ID NO: 10 shows the amino acid sequence of adenovirus 37 fiberprotein (x94484).

SEQ ID NO: 11 shows the nucleic acid sequence (V00383) encodingovalbumin.

SEQ ID NO: 12 shows the amino acid sequence of ovalbumin.

DETAILED DESCRIPTION

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference (including those so indicated). The provideddescription includes information that may be useful in understanding thepresent invention. It is not an admission that any of the informationprovided herein is prior art or relevant to the presently claimedembodiments, or that any publication specifically or implicitlyreferenced is prior art. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, example methods and materials aredescribed.

I. Abbreviations

Ab, antibody

AFC, Ab forming cells

Ag, antigen

CT, native cholera toxin

GALT, gut-associated lymphoreticular tissues

iLP, small intestinal lamina propria

MLNs, mesenteric lymph nodes

OVA, ovalbumin

OVA-pσ1, OVA genetically fused to protein sigma one of reovirus

PPs, Peyer's patches

S-IgA, secretory-IgA

Treg, regulatory T

II. Terms

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this disclosure belongs. Definitions of common terms in molecularbiology may be found in Benjamin Lewin, Genes V, published by OxfordUniversity Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments, the followingexplanations of specific terms are provided:

As used herein, the term “adjuvant” refers to a substance sometimesincluded in a vaccine formulation to enhance or modify theimmune-stimulating properties of a vaccine.

As used herein, the term “antibody” refers to a large Y shaped proteinmolecule made by B-cells of the immune system which very selectivelybinds to other specific protein molecules called antigens.

As used herein, the term “antigen” refers to a foreign substance thatthat when introduced into the body triggers an immune system response,resulting in production of an antibody as part of the body's defenseagainst disease.

As used herein, the term “DNA vaccine” refers to a eukaryotic expressionsystem encoding the molecular machinery for the expression of thesubunit vaccine encoded in plasmid nucleic acids.

As used herein, the term “expression” refers to the vaccine vector whichis responsible for producing the vaccine.

As used herein, the term “immunization” refers to a process by which aperson or animal becomes protected against a disease; the process ofinducing immunity by administering an antigen (vaccine) to allow theimmune system to prevent infection or illness when it subsequentlyencounters the infectious agent.

As used herein, the term “mucosal” means any membrane surface covered bymucous.

As used herein, “mucosal targeting ligand” refers to a viral protein oradhesins that specifically bind to the epithelia to enable uptake of thevaccine. These MTLs are not restricted to proteins, but can a proteinderivatized or not with carbohydrates and/or lipids. Likewise,carbohydrate, lipid, or nucleic acids found to bind to the epithelia canalso be included as mucosal targeting ligands. Methods for making MTLsand additional examples thereof are described in PCT/US2006/001346(published as WO 2006/078567), which is incorporated herein by referencein its entirety.

As used herein, the term “toleragen” means any antigen (such as aprotein, nucleic acid, carbohydrate, lipid, or combination of anythereof) that mediates host unresponsiveness. By way of example, atoleragen works by inducing the tolerized host not to produce antibodiesor cell-mediated immune responses specific for the toleragen. Additionaldiscussion of toleragens may be found, for instance, in PCT publicationWO 2006/052668, which is incorporated herein in its entirety.

III. Tolerizing Agents

One of the problems for conventional tolerization regimens is therequirement to use high doses, or repeated dosing, of antigen (toleragenor allergen). This disclosure provides evidence that the addition of atargeting molecule (or tolerizing agent), represented in variousembodiments by protein sigma 1 (pσ1), mediates tolerance after a singleoral dose or with minimal dosing. This enables use of far less toleragenwhen it is genetically fused to pσ1. As an example, typically, 25 mg oftoleragen (for instance, the test antigen used in this case, ovalbumin(OVA)) is required to be given twice orally in order to induce toleranceas measured by lack of proliferative T-cell responses to OVA, reducedanti-OVA antibody responses, and reduced delayed type hypersensitivityreactions. In contrast, a single, low oral dose (100 μg) of OVA-pσ1fusion protein was sufficient to elicit tolerance. This indicates thefusion is at least 500-fold more effective than convention.

Given this finding, the addition of a targeting molecule that directs(targets) a toleragen to the host M cells and/or mucosal epitheliumand/or host dendritic cells, mediates tolerance induction via binding tohost sialic acid, specific host receptors, or via a combination of theseor other mechanisms. Such binding events contribute in part or in wholeto the eventual development of tolerance.

In addition to pσ1, other ligands that contribute to binding to M cells,dendritic cells, and/or mucosal epithelium and thereby mediate toleranceto a passenger molecule are included. As example, adenovirus 35 fiberprotein or adenovirus 37 fiber protein, the latter of which has sialicacid binding activity and can also be used to elicit tolerance to amolecule fused or attached thereto. Any toleragen that can be fused tosuch (targeting) ligands, or adaption of such ligands for delivery ofparticles (e.g., nanoparticles, microspheres, liposomes, or virus-likeparticles), can be used to induce tolerance and thereby, for instance,prevent or treat autoimmune diseases, allergies, food allergies, orallow for tolerization to permit continued treatment with biologicals,e.g., botulinum neurotoxins (BoNTs).

Representative targeting molecules (or domains of molecules) thatcontribute to binding (e.g., to M cells, dendritic cells, and/or mucosalepithelium) include but are not limited to known viral proteins.Sequences of such proteins, and the nucleic acids encoding them, can befound in public databases, such as GenBank. For instance, in addition tospecific sequences discussed herein in detail, another nucleotidesequence encoding a human adenovirus 2 fiber protein is found underAccession No. AJ278923. Similarly, an example reovirus 3 signal is foundunder Accession No. X01161.

By way of example, the fusion of the pσ1 or like (tolerizing) moleculeto the heavy and/or light chain(s) of a BoNT allows the adaption of theresultant fusion protein as a prophylactic or therapeutic vaccine toprevent or treat immune reactivity against BoNT. BoNTs are currentlyused for a variety of treatments including tremor disorders.Consequently, repeated exposure to native BoNTs can result in thedevelopment of neutralizing antibodies to the BoNTs. Such exposure canprevent BoNT treatments. However, the use of a tolerizing molecule asdescribed, in conjunction with BoNT light and/or heavy chains, canprevent or treat this immune reactivity. Thus, this disclosure describesthe addition of mucosal targeting molecule(s) that enhance toleranceinduction.

One embodiment of this present disclosure is that certain molecules thatbind the mucosal epithelium can elicit tolerance in a subject. Thus, forexample, using the reovirus protein σ1, a subject can be “vaccinated”for instance nasally, orally, or peripherally for tolerance induction,thereby preventing the host (subject) from reacting against thepassenger antigen fused thereto. Evidence provided here shows thatOVA-pσ1, when given orally or nasally, makes the host unresponsive toOVA. In a similar fashion, when other proteins or peptides aregenetically engineered onto OVA—pσ1 or pσ1, tolerance to autoimmuneepitopes can also be induced. For example, peptides from mouseproteolipid protein or from myelin oligodendrocyte glycoproteingenetically engineered onto OVA-pσ1, when given, can reduce a multiplesclerosis-like disease. Thus, any components that induce human or animalautoimmune disease when fused to pσ1, and given to induce tolerance,should prevent or treat autoimmune diseases, such as multiple sclerosis,arthritis, diabetes, Hashimoto's disease, Graves' disease, Sjögrensyndrome, etc.

Another embodiment is that compounds described herein can be used toinduce tolerance to botulinum neurotoxins or other biologicaltherapeutic agents. Currently, botulinum neurotoxins are used to treattremor disorders as well as for cosmetic applications. However, oneside-effect is that the individual can develop neutralizing antibodiesresulting in the therapeutic loss of these treatments. Thus, an MTLfused to the β-trefoil vaccine, heavy chain, or the light chain tobotulinum neurotoxins. Thus, this shows that drugs or therapeutics canbe applied to pσ1 to limit the host response. These can also includehost inflammatory mediators, e.g., cytokines or soluble cytokinereceptors, such that the individual shows unregulated or elevatedexpression of these inflammatory mediators that need to be suppressed.

Also particularly contemplated are fusion proteins that contain atolerizing ligand (or its sialic acid binding domain component) thattargets the fusion protein to host M cells and/or mucosal epitheliumand/or host dendritic cells, and a component or fragment of at least onebotulinum neurotoxin from serotype A, B, C, D, E, F, or G that willinduce tolerance to botulinum. In some specific examples, the fusionprotein contains a component or fragment, or domain, from two or moreserotypes, or in some instances from all of serotypes A through G.

Tolerizing antigens include, but are not limited to, autoimmune antigens(“autoantigens”), therapeutically active biological agents, allergens,inflammatory antigens, and so forth. By way of example, therapeuticallyactive biological agents maybe any immunologically active (that is,immune stimulatory) proteins or peptides that have a therapeuticfunction, such as growth factors, hormones (e.g., insulin), clottingfactors (e.g., Factor VIII), metabolic enzymes, therapeutic antibodies(e.g., HERCEPTIN® or Trastuzumab), toxins (e.g., botulinum toxin), andso forth. Additional specific antigens that could usefully be fused to atargeting portion in the described fusion proteins will be known tothose of ordinary skill in the art. For instance, WO 2006/052668describes a number of representative antigens and categories thereofthat can be used for tolerization.

The fusion proteins described herein are useful as therapeutic compoundsfor treatment of subjects, including human and veterinary subjects. Asdemonstrated, routes of administration include oral and nasalapplication, though other routs are contemplated. The dosage form of apharmaceutical composition comprising one or more of the providedtolerizing fusion proteins will be influenced by the mode ofadministration chosen. For instance, in addition to injectable fluids,inhalational, topical, opthalmic, peritoneal, and oral formulations canbe employed. Inhalational preparations can include aerosols,particulates, and the like. In general, the goal for particle size forinhalation is about 1 μm or less in order that the pharmaceutical reachthe alveolar region of the lung for absorption. Oral formulations may beliquid (for example, syrups, solutions, or suspensions), or solid (forexample, powders, pills, tablets, or capsules). For solid compositions,conventional non-toxic solid carriers can include pharmaceutical gradesof mannitol, lactose, starch, or magnesium stearate. Actual methods ofpreparing such dosage forms are known, or will be apparent, to those ofordinary skill in the art.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets can be coated bymethods well known in the art. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups orsuspensions, or they can be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate.

For administration by inhalation, the compounds for use according to thepresent disclosure are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The pharmaceutical compositions that comprise at least one therapeuticagent, in some embodiments, will be formulated in unit dosage form,suitable for individual administration of precise dosages. The amount ofactive compound(s) administered will be dependent on the subject beingtreated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The therapeutically effective amount of therapeutic agent, andspecifically a tolerizing fusion protein, will be dependent on thespecific fusion protein utilized, the subject being treated, theseverity and type of the affliction, and the manner of administration.The exact dose is readily determined by one of skill in the art based onthe teachings herein, along with the potency of the specific compound,the age, weight, sex and physiological condition of the subject. By wayof example, in various embodiments the dosage of a tolerizing fusionprotein required to achieve (or maintain) tolerance in a subject is lowrelative to traditional tolerization regimens. For instance, as few asone or a few doses (e.g., fewer than about three, or fewer than aboutfive doses) of agent may be sufficient to induce tolerance. Similarly, arelatively low amount of antigen is required per dose, compared topreviously known tolerance approaches). By way of example, as little as1 mg or less of antigen in a dose (or total, in a series of doses) willbe effective with some fusion proteins. In other instances, as little as500 μg, 300 μg, 250 μg, or less in a dose, or total in a series ofdoses, or even as little as 200 μg, 150 μg, 100 μg, or less will beeffective. Based on, and the skill of practitioners who engage intolerance induction, specific dosages and dosage regimens can readily beworked out for any particular tolerizing fusion protein using theteachings herein.

Ovalbumin-Protein σ1 M Cell Targeting Enhances Oral Tolerance with Lossof OVA-Specific CD4⁺ T Cells

In this example, facilitated induction of oral tolerance using an Mcell-targeting protein antigen delivery system was examined. Mice werefed different doses of (1) a recombinant protein sigma one (pσ1) ofreovirus genetically conjugated to ovalbumin (OVA-pσ1) described hereinor (2) PBS prior to oral challenge with OVA plus cholera toxin asmucosal adjuvant. A low dose of OVA-pσ1 reduced anti-OVA antibody andCD4-positive (CD4⁺) T cell responses in both mucosal and systemiclymphoid tissues. OVA/MHC II-A^(d) tetramer staining revealed that thenumbers of OVA-specific CD4⁺ T cells were significantly more reduced inthe small intestinal lamina propria (iLP) of mice fed OVA-pσ1 than ofthose fed PBS, while no significant difference was seen for the spleen.The spleen of orally tolerized mice showed an increased frequency ofCD25⁺, CD4⁺ T cells with TGF-β1 production. These results show thatmucosal and systemic unresponsiveness are regulated by distinct T cellsubsets.

Experimental Procedures Mice

BALB/c mice were purchased from the Frederick Cancer Research facility(Frederick, Md.). Mice were housed in microisolators, maintained inhorizontal laminar flow cabinets, and provided sterile food and water aspart of a specific-pathogen-free facility in the Immunobiology VaccineCenter at the University of Alabama at Birmingham. The health of themice was monitored by both serology for bacterial and viral pathogensand immunohistology. All of the mice used in these experiments were freeof bacterial and viral pathogens.

Construction of OVA-pσ1 for M Cell Targeting

PCR was used to obtain the cloned pσ1 cDNA from reovirus serotype 3strain Dearing as previously described (Wu et al., Gene Ther. 7:61-69,2000). Ovalbumin (OVA) was genetically fused to pσ1s N-terminus and isreferred to as OVA-pσ1. The OVA-pσ1 was produced using a Pichia pastorisyeast expression system as a his-tag labeled protein.

Oral Immunization

Mice were gastrically intubated with different doses of OVA-pσ1dissolved in 0.25 ml of PBS. Control mice received PBS only. Seven dayslater, mice were orally immunized with 1 mg of OVA plus 15 μg of CTthree times at weekly intervals (Kato et al., J Immunol. 166:3114-3121,2001). OVA-specific T and B cell responses were determined seven daysafter the last immunization (Kato et al., J. Immunol. 166:3114-3121,2001).

OVA-Specific Antibody Assays

OVA-specific antibody (Ab) levels in plasma and mucosal secretions weredetermined by an ELISA as previously described (Kato et al., J. Immunol.166:3114-3121, 2001; Kato et al., Int. Immunol. 15:145-158, 2003;Fujihashi et al., Proc. Natl. Acad. Sci. (USA) 98:3310-3315, 2001;Hagiwara et al., J. Immunol. 170:1754-1762, 2003; Kataoka et al., J.Immunol. 172:3612-3619, 2004). Briefly, 96-well FALCON™ microtest assayplates (BD BioSciences, Oxnard, Calif.) were coated with one mg/ml ofOVA in PBS. After blocking with 1% BSA in PBS, two-fold serial dilutionsof samples were added to each well. Following incubation overnight at 4°C., horseradish peroxidase (HRP)-labeled goat anti-mouse μ, γ or α heavychain-specific Abs (Southern Biotechnology Associates (SBA), Birmingham,Ala.) were added to wells. The color reaction was developed for fifteenmin at room temperature with 100 μl of 1.1 mM 2, 2′-azino bis(3-ethylbenz-thiazoline-6-sulfonic acid) in 0.1 M citrate phosphatebuffer (pH 4.2) containing 0.01% H₂O₂. Endpoint titers were expressed asthe reciprocal log₂ of the last dilution that gave an optical density at415 nm of 0.1 greater than background.

Lymphoid Cell Isolation and Enumeration of Ab-Forming Cells

The spleen and MLNs were removed aseptically and single-cell suspensionsprepared in RPMI 1640 (Cellgro Mediatech, Washington, D.C.) containingHEPES buffer, non-essential amino acids, sodium pyruvate, L-glutamine,penicillin, streptomycin and gentamycin (incomplete medium) by passagethrough sterile wire mesh screens as described previously (Kato et al.,J. Immunol. 166:3114-3121, 2001; Fujihashi et al., Proc. Natl. Acad.Sci. (USA) 98:3310-3315, 2001). Peyer's patches (PPs) were carefullyexcised from the small intestinal wall and dissociated using the neutralprotease enzyme collagenase type IV (Sigma) in incomplete RPMI 1640 toobtain single-cell preparations (Kato et al., J. Immunol. 166:3114-3121,2001, Kato et al., Int. Immunol. 15:145-158, 2003). Mononuclear cells inthe iLP were isolated after removal of PP and intraepitheliallymphocytes from the small intestine using a combination of enzymaticdissociation and discontinuous PERCOLL™ density gradients (PharmaciaFine Chemicals, Uppsala, Sweden). Mononuclear cells in the interfacebetween the 40% and 75% layers were removed, washed and resuspended inRPMI 1640 containing 10% FCS (complete RPMI 1640) (Kato et al., J.Immunol. 166:3114-3121, 2001; Fujihashi et al., Proc. Natl. Acad. Sci.(USA) 98:3310-3315, 2001). Mononuclear cells obtained from mucosal andsystemic lymphoid tissues were subjected to an ELISPOT assay in order todetect numbers of OVA-specific Ab-forming cells (AFCs) (Kato et al., J.Immunol. 166:3114-3121, 2001; Kato et al., Int. Immunol. 15:145-158,2003; Fujihashi et al., Proc. Natl. Acad. Sci. (USA) 98:3310-3315, 2001;Fujihashi et al., J. Exp. Med. 183:1929-1935, 1996; Hagiwara et al., J.Immunol. 170:1754-1762, 2003; Kataoka et al., J. Immunol. 172:3612-3619,2004). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore,Bedford, Mass.) were coated with one mg/ml of OVA for analysis ofanti-OVA-specific AFCs. The numbers of OVA-specific AFCs were quantifiedusing an IMMUNOSPOT® spot analyzer (Cellular Technology Ltd., Cleveland,Ohio) (Hagiwara et al., J. Immunol. 170:1754-1762, 2003; Kataoka et al.,J. Immunol. 172:3612-3619, 2004).

Delayed Type Hypersensitivity (DTH) Responses

OVA-specific DTH responses were measured 7 days after the last oralchallenge with OVA plus CT, as described above. Briefly, PBS (20 μl)containing 10 μg of OVA was injected into the left ear pinna of micewhile the right ear pinna received a PBS control injection (Kato et al.,J. Immunol. 166:3114-3121, 2001; Fujihashi et al., Acta. Odontol. Scand.59:301-308, 2001; Kato et al., Int. Immunol. 15:145-158, 2003). Earswelling was measured 24 hours later with a dial thickness gauge (OzakiManufacturing Co., Ltd., Tokyo, Japan). The DTH response was expressedas the increase in ear swelling after OVA injection minus the swellingin the PBS-injected control site.

Ag-Specific T Cell Responses

CD4⁺ T cells from spleen, MLNs, and PPs were purified by use of anautomated magnetic activated cell sorter (AUTOMACS™) system (MiltenyiBiotec, Auburn, Calif.), as described previously (Hagiwara et al., J.Immunol. 170:1754-1762, 2003; Kataoka et al., J. Immunol. 172:3612-3619,2004). Briefly, a nylon wool column of an enriched T cell fraction wasincubated with biotinylated anti-CD4 mAb (GK 1.5) (BD PharMingen)followed by streptavidin-conjugated microbeads and sorted to purity withthe AUTOMACS™. This purified T cell fraction was >97% CD4⁺ and the cellswere >99% viable. The purified CD4⁺ T cell fraction was then resuspendedin complete RPMI 1640 (4×10⁶ cells/ml) and cultured in the presence ofone mg/ml OVA of cultures of T cell-depleted, irradiated (3000 rad)splenic antigen-presenting cells taken from non-immunized, normal mice.To assess OVA-specific T cell proliferative responses, an aliquot of 0.5μCi of tritiated [³H]-TdR (Amersham Biosciences, Arlington Heights,Ill.) was added during the final 18 hour of incubation, and the amountof [³H]-TdR incorporation was determined by scintillation counting. Thesupernatants of identically treated T cell cultures not incubated with[³H]-TdR were then subjected to a cytokine-specific ELISA as describedbelow.

Cytokine-Specific ELISA

Levels of cytokines in culture supernatants were measured by an ELISA.The details of the ELISA for IFN-β, IL-2, IL-4, IL-5, IL-6 and IL-10have been described previously (Kato et al., J. Immunol. 166:3114-3121,2001; Kato et al., Int. Immunol. 15:145-158, 2003; Fujihashi et al.,Proc. Natl. Acad. Sci. (USA) 98:3310-3315, 2001; Hagiwara et al., J.Immunol. 170:1754-1762, 2003; Kataoka et al., J. Immunol. 172:3612-3619,2004). The following were used as coating and detection mAbs,respectively: anti-IFN-β: R4-6A2 and XMG 1.2 mAbs; anti-IL-2: JES6-1A12and JES6-5H4 mAbs; anti-IL-4: BVD4-1D11 and BVD6-24G2 mAbs; anti-IL-5:TRFK-5 and TRFK-4 mAbs; anti-IL-6: MP5-20F3 and MP5-32C11 mAbs; andanti-IL-10: JES5-2A5 and JES5-16E3 mAbs. A mouse TGF-β1 immunoassay kit,QUANTIKINE™ M (R & D systems, Minneapolis, Minn.), was used to detectTGF-β1 in the culture supernatants. The levels of Ag-specific cytokineproduction were calculated by subtracting the results of controlcultures (e.g., without Ag stimulation) from those of Ag-stimulatedcultures. This ELISA was capable of detecting 0.8 ng/ml of IFN-β; 0.4U/ml of IL-2; 25 pg/ml of IL-4; 0.8 U/ml of IL-5; 200 pg/ml of IL-6; 4pg/ml of IL-10; and 4 pg/ml of TGF-β31.

Flow Cytometry Sorting and Analysis

In order to determine the frequencies of OVA-specific CD4⁺ T cells,mononuclear cells from spleen, MLNs, PPs and iLP were stained withFITC-conjugated anti-CD4 (GK1.5), biotinylated anti-CD25 (7D4) mAb andPE-labeled OVA/MHC II-A^(d) tetramer followed by Cy5.5-streptavidinbefore being subjected to flow cytometric analysis. For intracellularIL-10 analysis, cells were incubated with ionomycin (1 μg/ml, SIGMA, St.Louis, Mo.) and phorbol 12-myristate 13-acetate (PMA, 25 ng/ml, SIGMA)for 6 hours and then stained with PE-labeled anti-CD4, biotinylatedanti-CD25 mAbs followed by Cy5.5-streptavidin. These samples werefurther stained intra-cellularly with ALEXA FLUOR® 488 labeledanti-IL-10 mAb (JES5-16E3). In some experiments, cells were stained withFITC-labeled anti-CD4 and biotinylated anti-CD25 mAb followed byPE-streptavidin. CD4⁺ CD25⁺ T cells were purified by flow cytometry andtheir TGF-β1 production was determined as described above.

Statistics

The significance of the difference (e.g., p values) among groups wasevaluated by the Mann Whitney U test using a Statview II programdesigned for Macintosh computers.

Results Optimization of Oral Doses of OVA-pσ1

Since it has been shown that pσ1 can bind to mucosal M cells (Wu et al.,Proc. Natl. Acad. Sci. (USA) 98:9318-9323, 2001), it can be hypothesizedthat oral tolerance can be effectively achieved by OVA-pσ1. To test thisnotion, mice were gastrically intubated with different doses of OVA-pσ1.Mice were fed one dose of either 100 μg, 500 μg or 1000 μg of OVA-pσ1.An additional group of mice was given three daily doses of 100 μg oforal OVA-pσ1. Seven days later, all groups of mice were challenged oncea week for three weeks with oral OVA plus CT. OVA-specific plasma IgG Abtiters were not markedly reduced in mice given three weekly doses of 100μg of OVA-pσ1 (FIG. 1). On the other hand, they were significantly morereduced in all other single OVA-pσ1 treatment groups than in mice fedPBS (FIG. 1). Further, OVA-specific plasma IgA and mucosal S-IgA Abresponses in mouse groups receiving one feeding of OVA-pσ1 were markedlylower than in the positive control group (PBS-fed mice) (FIG. 1). Theseresults show that a single oral dose of OVA-pσ1 effectively induces bothsystemic and mucosal unresponsiveness to OVA. Based upon these results,we next employed a single oral dose of 100 μg of OVA-pσ1 for furtherexperiments.

Oral OVA-pσ1 Facilitates Both Systemic and Mucosal Unresponsiveness

To further confirm these findings at the cellular level, the numbers ofOVA-specific Ab-forming cells (AFCs) were examined in various lymphoidtissues of mice given oral OVA-pσ1 or PBS. Numbers of OVA-specific IgGand IgA AFCs in spleen and mesenteric lymph nodes (MLNs) were reducedsignificantly (p<0.05) in the oral pσ1-group but not in the oralPBS-Group (FIG. 2), showing that oral tolerance is indeed induced byfeeding 100 μg of OVA-pσ1. In order to assess induction ofunresponsiveness in mucosal effector sites, the numbers of OVA-specificAFCs in iLP were compared in groups fed OVA-pσ1 or PBS. The number ofanti-OVA IgA AFCs was reduced in the pσ1—but not the PBS-fed group (FIG.2). These results suggest that M cell targeting by OVA-pσ1 effectivelyinduces mucosal tolerance and may contribute to the maintenance ofmucosal homeostasis.

DTH and CD4⁺T Cell Proliferative Responses

Whether tolerance was induced at the T cell level after a single oraldose of OVA-pσ1 was next determined. OVA-specific delayed-typehypersensitivity (DTH) responses were assessed in mice given eitherOVA-pσ1 or PBS orally. OVA-specific DTH responses were much morepronounced in the pσ1-group than in the PBS group (FIG. 3A), showingthat OVA-specific T cell responses were tolerized by a single low doseof OVA-pσ1. Using the described oral challenge system, which allowsexamination of CD4⁺ T cell responses in mucosal lymphoid tissues, CD4⁺ Tcell proliferative responses were next examined in both mucosal (MLNsand PPs) and systemic (spleen) compartments of mice given oral OVA-pσ1.The CD4⁺ T cells from spleen, PPs, and MLNs were purified by use of anautomated magnetic-activated cell sorter (AUTOMACS™) system. Thesepurified CD4⁺ T cell fractions were cultured with or without one mg/mlof OVA in the presence of T cell-depleted, irradiated splenic APCs takenfrom non-immunized, normal mice. Significant reductions in T cellproliferative responses were seen in the spleen, MLNs and PPs of theOVA-pσ1—but not the PBS-fed group (FIG. 3B). These results show that Tcell unresponsiveness was initiated in mucosal inductive tissues such asthe PPs, by M cell targeting of OVA-pσ1. Subsequently, these tolerizedCD4⁺ T cells migrated into the spleen via the MLNs.

Cytokine Production by OVA-Stimulated CD4⁺ T Cells

Since T cell unresponsiveness was induced in both systemic and mucosallymphoid tissues by a single oral dose of OVA-pσ1, Th1- and Th2-typecytokine production by OVA-stimulated CD4⁺ T cells was examined.Purified CD4⁺ T cells from the spleen and PPs of mice fed OVA or PBSwere incubated with or without 1 mg of OVA in the presence of autologousAPCs for five days. When the culture supernatants were harvested andexamined by cytokine-specific ELISA, OVA-pσ1-fed mice showed reducedCD4+ Th1 (IFN-γ and IL-2) and Th2 (IL-4, IL-5, IL-6 and IL-10) cytokineresponses, while mice fed oral PBS showed high levels of Th2-typecytokines, especially IL-4 and IL-10 (Table 1). A virtually identicalprofile of up-regulation of Th2-type cytokine synthesis was seen in thespleen of mice following oral administration of PBS. On the other hand,a hyporesponsive Th1- and Th2-type cytokine profile was noted in bothPPs and spleen of mice fed OVA-pσ1 before being orally challenged withOVA plus CT (Table 1). Taken together, these results indicate that CD4⁺T cell unresponsiveness was induced in both spleen and PPs by a singleoral dose of OVA-pσ1.

TABLE 1 CD⁺ Th1 and Th2 Cytokine Synthesis by OVA-Specific CD4⁺ TCells^(a) Orally Th1 type^(b) Th2 type^(b) Lymphoid Immunized IFN-γ IL-2IL-4 IL-5 IL-6 IL-10 Tissue with (ng/ml) (ng/ml) (pg/ml) (ng/ml) (ng/ml)(ng/ml) Spleen PBS 5.9 ± 1.3^(c)  0.9 ± 0.18 477 ± 2.6 4.54 ± 0.2 1.28 ±0.05 44.5 ± 2.9 OVA-pσ1 0.3 ± 0.02^(e) 0.19 ± 0.02^(f)  30 ± 0.8^(e)0.18 ± 0.02^(e) 0.07 ± 0.02^(e)  1.8 ± 0.3^(e) Peyer's PBS 4.2 ± 1.7 1.6 ± 0.05 420 ± 3.0  3.1 ± 0.2  0.8 ± 0.08 40.8 ± 1.1 Patches OVA-pσ10.3 ± 0.03^(e) 0.15 ± 0.01 110 ± 1.1^(f) 0.27 ± 0.02* 0.12 ± 0.01^(f) 2.2 ± 0.2^(e) ^(a)Splenic CD4+ T cells (2 × 10⁶/ml) from each group ofmice were cultured with 1 mg/ml of OVA in the presence of Tcell-depleted and irradiated splenic feeder cells (4 × 10⁶/ml).^(b)Culture supernatants were harvested after 5 days (2 days for IL-2)of incubation and analyzed by the cytokine-specific ELISA. ^(c)Theresults represent the mean ± one SEM of one of three separateexperiments. ^(d)N.D. indicate not detected. ^(e)ρ < 0.01, ^(f)ρ < 0.05compared with PBS group.

Mucosal Unresponsiveness is Due to Clonal Deletion of OVA-Specific CD4⁺T Cells

In order to examine the role of OVA-specific CD4⁺ T cells in oraltolerance, mononuclear cells from spleen, PPs, MLNs and iLP wereisolated one week after the last immunization and stained withFITC-conjugated anti-CD4, biotin-conjugated anti-CD25 mAbs andPE-labeled OVA/II-A^(d) tetramer followed by Cy5.5-streptavidin. Thisanalysis revealed a lower frequency of tetramer⁺ OVA-specific CD4⁺ Tcells in iLPs of mice given OVA-pσ1 prior to oral challenge with OVAplus CT than in mice given oral PBS (FIG. 4 and Table 2). Numbers ofOVA-specific CD4⁺ T cells were reduced in PPs and MLNs of mice givenoral OVA-pσ1 (Table 2), but remained essentially the same in the spleenof orally tolerized mice and of those exhibiting high OVA-specific Abtiters (Table 2). When CD25 expression on tetramer⁺ OVA-specific CD4⁺ Tcells was examined, the frequency of CD4⁺ CD25⁺ T cells was found to besignificantly decreased in iLP of orally tolerized mice (Table 2). Inaddition, the numbers of tetramer⁺ OVA-specific CD4⁺ CD25⁻ T cells inspleen, MLNs, PPs and iLP were also significantly reduced. Among theselymphoid tissues, marked reductions in OVA-specific CD4⁺ CD25⁻ T cellswere seen in the iLP of orally tolerized mice (Table 2). On the otherhand, increased numbers of CD4⁺ CD25⁺ T cells, especially of theOVA/II-A^(d) tetramer negative subset, were noted in spleen and MLNs ofmice give oral OVA-pσ1 before mucosal challenge with OVA plus CT (Table2). These results suggest that mucosal unresponsiveness to orallydelivered Ag is most likely due to the reduced numbers of OVA-specificCD4⁺ T cells in the iLP (clonal deletion), a mechanism that is entirelydistinct from the systemic unresponsiveness induced by activesuppression by CD4⁺ CD25⁺ Treg cells.

TABLE 2 The frequency of OVA-specific CD4⁺ T cells in various lymphoidtissues^(a.) CD4⁺ (100%) Orally OVA/I-A^(d) OVA/I-A^(d) OVA/I-A^(d)Lymphoid Immunized Tetramer⁺ Tetramer⁺ Tetramer⁻ Tissue With CD25⁺ CD25⁺CD25⁺ Spleen PBS 4.6 ± 0.4 5.1 ± 0.5 8.2 ± 0.4 OVA-pσ1 5.6 ± 1.1  3.6 ±0.6^(c) 12.0 ± 0.8  MLNs PBS 1.6 ± 0.3 2.8 ± 0.1 5.6 ± 1.1 OVA-pσ1 1.8 ±0.3  2.0 ± 0.2^(b)  8.6 ± 1.0^(d) Peyer's PBS 2.6 ± 0.3 5.9 ± 0.6 6.3 ±1.8 patches OVA-pσ1 2.7 ± 0.7 4.2 ± 0.5 5.2 ± 1.1 Intestinal PBS 1.9 ±0.3 2.7 ± 0.2 4.5 ± 0.4 lamina OVA-pσ1  0.8 ± 0.2^(b)  1.4 ± 0.1^(b) 2.9± 0.4 propria ^(a)Mononuclear cells (1 × 10⁶) from various lymphoidtissues of mice fed OVA-pσ1 or PBS were stained with FITC-conjugatedanti-CD4 (GK 1.5) and biotinylated anti-CD25 (7D4) mAbs as well asPE-labeled OVA/I-A^(d) tetramer followed by Cy5.5-streptavidin. Sampleswere then subjected to flow cytometry analysis using FASCALIBUR ™. Theresults represent the mean values ± one SEM from these separateexperiments. ^(b)p < 0.01. ^(c)p < 0.03 ^(d)p < 0.05 compared withPBS-group.

TGF-β1-Producing Treg Cells are Induced by Oral OVA-pσ1

The increased frequency of CD4⁺ CD25⁺ T cells in spleen and MLNssuggested the possibility that CD4⁺ Treg cells are induced when mice arefed OVA-pσ1 and then mucosally challenged with OVA plus CT as mucosaladjuvant. To test this possibility, we examined the production of IL-10and TGF-β1 by CD4⁺ CD25⁺ T cells. Flow cytometry-purified CD4⁺ CD25⁺ Tcells from PPs, spleen and MLNs of mice fed OVA-pσ1 or PBS werestimulated with OVA for 5 days. The culture supernatants of CD4³⁰ CD25⁺T cells from orally tolerized mice contained higher levels of TGF-β1than did those from PBS-fed mice (FIG. 5A). Intracellular IL-10 analysiswas performed to determine the extent of IL-10 production by CD4⁺ CD25⁺Treg cells in spleen and MLNs of orally tolerized mice. Flow cytometricanalysis revealed fewer IL-10-producing CD4⁺ CD25⁺ T cells in mice fedOVA-pσ1 than in mice fed PBS (FIG. 5B). These results demonstrate thatTGFβ1-producing CD4⁺ Treg cells were induced in the MLNs and spleen ofmice fed OVA-pσ1.

Discussion

The current study shows that the OVA-pσ1 M cell-targeting deliverysystem facilitates the induction of oral tolerance. Mucosal and systemicunresponsiveness can be induced with a single oral dose of 100 μg ofOVA-pσ1 instead of the repeated low doses of oral OVA that wouldotherwise be required. OVA-specific mucosal S-IgA and plasma IgG Abresponses as well as DTH and T cell proliferative responses were allreduced significantly in OVA-pσ1—but not in PBS-fed mice. Further,OVA-stimulated CD4⁺ T cells from spleen and PPs of orally tolerized miceshowed much more marked reduction in the levels of both Th1- andTh2-type cytokine production than did those fed PBS before being orallychallenged with OVA plus CT as adjuvant. The use of OVA/MHC II-A^(d)tetramer staining revealed significantly reduced numbers of OVA-specificCD4⁺ T cells in iLP of mice fed OVA-pσ1. On the other hand, the numbersof TGF-β1-producing CD4⁺ CD25⁺ T cells were higher in the MLNs andspleen of orally tolerized mice than in the control group. These resultsshow that the M cell-targeting Ag delivery by OVA-pσ1 feedingeffectively induces mucosal and systemic unresponsiveness. Of keyimportance is the finding that the mechanisms regulating tolerance inmucosal and peripheral lymphoid tissues are distinct.

The M cells are known to take up and transport lumenal Ags, includingproteins, viruses, bacteria, small parasites, and microspheres (Ermak etal., Cell Tissue Res. 279:433-436, 1995; Neutra et al., Cell 86:345-348,1996; Gebert et al., Int. Rev. Cytol. 167:91-159, 1996; Wolf & Bye,Annu. Rev. Med. 35:95-112, 1984). M cells have then been shown todeliver the intact Ag into underlying lymphoid tissue of the GALT(Gebert et al., Int. Rev. Cytol. 167:91-159, 1996; Wolf & Bye, Annu.Rev. Med. 35:95-112, 1984). M cells are also thought to be involved inAg processing and presentation, since the GALT M cells express MHC classII molecules and acidic endosomal-lysosomal compartments (Allan et al.,Gastroenterology 104:698-708, 1993). In addition to serving as a meansof transport for lumenal Ags, the M cells also provide an entryway forpathogens. For example, invasive strains of Salmonella typhimuriuminitiate murine infection by invading the M cells of the PPs (Jones etal., J. Exp. Med. 180:15-23, 1994). Based upon these findings, Mcell-targeting Ag delivery could be assumed to be the normal pathway forinduction of Ag-specific immune responses. Indeed, NALT M cell targetinga DNA vaccine constructed with pσ1 elicited Ag-specific IgG and S-IgA Abresponses (Wu et al., Proc. Natl. Acad. Sci. (USA) 98:9318-9323, 2001).However, our current study has now shown that oral administration ofOVA-pσ1 facilitates unresponsiveness to OVA in both systemic and mucosallymphoid tissues instead of inducting OVA-specific immunity. Theseopposite outcomes can be partially explained by the nature of the Ag.Ovalbumin is only weakly immunogenic and always requires an adjuvant forinduction of immune responses. In contrast, cytomegalovirus plasmid DNA(pCMV), a known ligand for toll-like receptor 9, is recognized by IFN-γproducing cells and dendritic cells (Krug et al., Immunity 21:107-119,2004) and most likely induces innate and acquired immunity. Indeed,although M cells are able to transport lumenal Ags, noninvasive strainsof S. typhimurium cannot penetrate M cells and are avirulent (Jones etal., J. Exp. Med. 180:15-23, 1994). An antigen's immunogenicity andpathogenicity in the GI tract could be the most critical factors indetermining whether mucosal immunity or tolerance is induced.

Mucosal tolerance may be the most common immune response because it isnecessary to maintain homeostasis. The normal host would readilyestablish unresponsiveness to commensal bacteria, food Ag and allergens.Taken together, we conclude that our OVA-pσ1 system, M cell targeting ofa non-pathogenic protein Ag is an efficient strategy for theestablishment of oral tolerance.

Results provided herein clearly show that M cell targeting Ag deliverysystem reduced the doses of feeding Ag in order to establish oraltolerance. Similar findings were reported using Ag conjugated with Bsubunit of CT (CT-B) (Sun et al., Proc. Natl. Acad. Sci. (USA)91:10795-10799, 1994). That study showed that a single oraladministration of relatively small amounts of particulate or solubleantigen coupled to the CT-B markedly suppressed systemic immuneresponses (Sun et al., Proc. Natl. Acad. Sci. (USA) 91:10795-10799,1994). Since CT-B specifically bind to GM-1 ganglioside which abundantlyexpressed by intestinal epithelial cells (iECs) including M cells, itstill remained unclear which of iECs or M cells play a significant rolein the induction of oral tolerance. The findings reported herein showedthat induction of oral tolerance can be easily achieved by M celltargeting Ag delivery system most likely without Ag uptake from iECs.Recent studies showed that M cells are present in the small intestine ofisolated lymphoid follicles (ILFs) as well as intestinal villi (villousM cells) (Hamada et al., J. Immunol. 168:57-64, 2002; Jang et al., Proc.Natl. Acad. Sci. (USA) 101:6110-6115, 2004). Role(s) of M cells in thesenewly identified GALT in the induction of oral tolerance are beinginvestigated.

Flow cytometric analysis revealed increased numbers of CD4⁺ CD25⁺ Tcells in MLNs and spleen of orally tolerized mice, suggesting feedingwith OVA-pσ1 induced production of Treg cells. Along this line, a recentstudy reported that PP-derived Treg clones produce high levels of TGF-β1and suppressed Ag-specific Ab responses in spleen (Tsuji et al., Int.Immunol. 15:525-534, 2003). Based upon these findings, our groupexamined TGF-β1 and IL-10 production by OVA-specific CD4⁺ T cells frommice fed OVA-pσ1 prior to oral challenge with OVA plus CT. Our resultsclearly show that CD4⁺ CD25⁺ T cells in PPs, MLNs and spleen from orallytolerized mice produce higher levels of TGF-β1 after OVA stimulationthan do those from mice fed PBS. On the other hand, intracellular IL-10production by CD4⁺ CD25⁺ T cells from mice fed OVA-pσ1 was significantlyreduced. Taken together with the observation that acquired-type CD4⁺Treg cells are Ag-specific and produce inhibitory cytokines includingTGF-β1 and IL-10 (Cottrez & Groux, Transplantation 77:512-15, 2004), ourresults indicate that acquired-type CD4⁺ Treg cells are induced by oraladministration of OVA-pσ1.

OVA-specific CD4⁺ T cells were significantly more reduced in the iLP oforally tolerized mice than in PBS-fed mice challenged with oral OVA plusCT, but no such reduction was seen in spleen or MLNs of either group.Similarly, others showed that a reduction of Ag-specific T cellsoccurred in mice given repeated low doses of cytochrome c protein(Gutgemann et al., Immunity 8:667-673, 1998). In contrast, the spleen oforally tolerized mice exhibited increased numbers of CD4⁺ CD25⁺ T cellsand the presence of TGF-β1-producing CD4⁺ CD25⁺ T cells. Based uponthese findings, it appears likely that mechanisms for the induction ofmucosal and systemic unresponsiveness differ. Thus, mucosalunresponsiveness is clearly associated with clonal deletion ofOVA-specific effector CD4⁺ T cells while systemic unresponsiveness maybe achieved by active suppression of an acquired type of Treg cells.These findings are the first to show that two separate mechanismsunderlie mucosal unresponsiveness and that they are entirely distinctfrom those which underlie systemic unresponsiveness.

It still remains unclear how this clonal deletion of OVA-specific CD4⁺ Tcells actually occurs since CD4⁺ CD25⁺ T cells are also reducedsignificantly in the iLP of orally tolerized mice. However, one canhypothesize that the numbers of OVA-specific CD4⁺ T cells and AFC in iLPare reduced simply because OVA-specific CD4⁺ T cell migration into theiLP has been interrupted. Thus, effector CD4⁺ Th cells could besuppressed by PP-derived TGF-β1-producing CD25⁺ Treg cells in the MLNsand spleen before reaching the iLP. To support this view, our previousresults showed that induction of Ag-specific Ab responses in the iLPsrequired three consecutive weekly oral immunizations (Kato et al., J.Immunol. 166:3114-3121, 2001; Fujihashi et al., J. Exp. Med.183:1929-1935, 1996). We are currently testing this notion using a nasalchallenge system in order to better distinguish between OVA-specificCD4⁺ effector T cell and CD4⁺ Treg cell activities.

In summary, this example provides the first evidence that M celltargeting of a non-pathogenic Ag OVA-pσ1 can induce mucosalunresponsiveness via a mechanism distinct from that underlying systemictolerance. This M cell-targeting system allowed us to elucidate theimmunoregulatory mechanisms of the PP-mediated oral tolerance pathwayfrom other potential mechanisms. Thus, these findings show thatregulatory-type CD4⁺ T cells are induced in the PP and then migrate intoMLNs and spleen. These CD4⁺ Treg cells contribute to the successfulsystemic unresponsive state that ensues. Further, these results clearlyshow that mucosal unresponsiveness to orally administered Ag can beattributed to a lack of Ag-specific CD4⁺ T helper cells in the iLP.

Nasal Tolerance

From the literature, it has been shown that reovirus type 3 proteinsigma 1 (pσ1) is a highly structured protein featuring several domains,which mediate a multi-step interaction between the virus and the hostcell (Barton et al., J. Biol. Chem. 276:2200-2211, 2000; FIG. 6). It hasbeen shown that type 3 pσ1 interacts with at least two host receptorsvia separate binding domains. The head domain binds with a component oftight junctions, JAM-1 molecule, whereas sequences contained within thefibrous tail domain binds terminal α-linked sialic acid residues on hostcells (Barton et al., J. Biol. Chem. 276:2200-2211, 2000; Chappell etal., J. Virol. 71:1834, 1997). To determine the relevant bindingcomponents of our recombinant pσ1 and OVA-pσ1, additional constructs orvariants were made and expressed in yeast (FIG. 6).

To determine the role of pσ1's sialic binding domain (SABD), a pσ1 (m)construct was made in which the mutations N198→D198 and R202→G202 wereintroduced to interrupt the SABD's binding activity. In addition, OVAwas genetically fused to pσ1(m) and called OVA-pσ1(m). Genetic fusionsof OVA are all placed at the N-terminus of pσ1 so as to not interferewith the host receptor binding domains located in the pσ1's C-terminus.Thus, if sialic acid binding dictates mediation of tolerance by pσ1,then the loss of sialic acid binding should confer immunization. In asimilar fashion, the complete removal of pσ1's SABD should do the same,and this variant, OVA-pσ1(Δ), which encompasses the OVA gene fused tothe last 207 amino acids of pσ1 renders only a functional trimerizingdomain and head (FIG. 6). Each of the OVA fusion proteins featured aflexible linker between the fusion partners.

Sialic Acid Binding is Important for Tolerance Induction by Pσ1

To determine the functional consequence of sialic acid binding byOVA-pσ1, groups of C57BL/6 mice were given three nasal immunizations ondays 0, 7, and 14 in combination with the mucosal adjuvant, choleratoxin (CT), and one of three antigens, OVA-pσ1, OVA-pσ1(Δ), or OVA orgiven OVA without CT. Again, OVA-pσ1(Δ) is a truncated OVA-pσ1 lackingits SABD and shaft (FIG. 6). To test for a delayed-type hypersensitivity(DTH) reaction, mice were challenged after 42 days with OVA into one earpinna and PBS into the other ear pinna. Mice immunized with OVA alone orOVA-pσ1+CT failed to show swelling in the OVA-challenged ear whencompared to mice immunized with OVA+CT (P<0.001) or OVA-pσ1(Δ)+CT(P=0.002) (FIG. 7). Thus, the OVA-pσ1 (Δ), which lacked the SABD,behaved more as an immunogen in contrast to OVA-pσ1, which behaved as atoleragen. This collective evidence suggested that the presence of theSABD on pσ1 was required for tolerance induction, whereas, in itsabsence, clearly immunization occurred.

Adoptive Transfer of CD4⁺ T Cells into Naive Mice Are Unresponsive toOVA Challenge

To test whether nasal exposure to OVA-pσ1 could make CD4⁺T cellsunresponsive to OVA and effectively adoptively transfer these T cells,the transgenic D011.10 CD4⁺ T cells were isolated from spleen and lymphnodes by cell-sorting, and adoptively transferred into naive BALB/cmice. After 24 hours, groups of mice were dosed nasally with PBS, 80 μgOVA-pσ1, or 400 μg OVA, or given a single i.m. OVA immunization. Threedays later, cervical lymph nodes (CLN) were removed and CD4⁺ T cellswere isolated by cell-sorting. These CLN CD4⁺ T cells (2×10⁶/mouse) wereadoptively transferred into naive mice, and after 24 hrs, they werechallenged s.c. with OVA in incomplete Freund's adjuvant. Five dayslater, CD4⁺ T cells from the head and neck LN (HNLN) were isolated bycell-sorting and cultured with mitomycin C-treated feeder cells withoutand with 1.0 mg OVA for 5 days. ³H-TdR was used to measure T cellproliferation. Mice were made unresponsive by the nasal 400 μg OVA orthe 80 μg OVA-pσ1 since these did not proliferate (FIG. 8). In contrast,the CD4⁺ T cells isolated from the i.m. OVA-dosed mice were responsive.Thus, dosing i.n. with OVA-pσ1 can make mice unresponsive to OVA, andthis unresponsiveness is mediated by CD4⁺ T cells specific for OVA.Moreover, this unresponsiveness can be adoptively transferred with CD4⁺T cells.

OVA-pσ1 can be Modified with Encephalitogenic Peptides to RenderProtection Against Experimental Autoimmune Encephalitis (EAE) Challenge

Thus far, we showed the feasibility of inducing tolerance against OVA, afamiliar antigen frequently used in experimental systems. To forwardefforts to treating autoimmune diseases, we adapted the OVA-pσ1 fusionprotein with peptides known to cause autoimmune disease. We hypothesizedthat genetic fusion of encephalitogenic peptides to OVA-pσ1 shouldinduce tolerance as shown with our studies using OVA as a test antigen.OVA-pσ1 was modified because we could then follow unresponsiveness toOVA as an internal control for our studies. Thus, this modified OVA-pσ1construct, termed AR1, was made with two copies of the encephalitogenicpeptide from proteolipid protein (PLP), PLP₁₃₉₋₁₅₁, separated by anirrelevant peptide (MOG₃₅₋₅₅) (FIG. 9A). C57BL/6 mice were dosed thricewith AR1, and they did not generate IgG or IgA anti-OVA Abs whencompared to OVA+CT-dosed mice (FIG. 9B-D). Subsequent to i.n. challengewith OVA+CT and then tested for a DTH response, no DTH reactions weredetected when compared to OVA+CT-dosed mice (FIG. 9E). A separate groupof mice also was orally fed AR1 and peripherally challenged with OVA+CT,and these too were unresponsive in this DTH assay. Thus, themodification of OVA-pσ1 with encephalitogenic peptides did not interferewith its ability to elicit OVA tolerance.

To test whether tolerance to the fused encephalitogenic peptides wasinduced by evaluating the efficacy of AR1 against PLP₁₃₉₋₁₅₁ challenge,SJL/J mice were nasally given AR1 as described in FIG. 9. For a positiveoral tolerance control group, myelin basic protein was given seven timesevery 2 days over a 2-wk course. As a negative control group, mice weredosed with PBS. Three wks after the onset of treatments, mice werechallenged s.c. with emulsified PLP₁₃₉₋₁₅₁ following standard protocols,and pertussis toxin (PT) was given i.p. A second PT dose was given twodays later. Following this challenge protocol, mice typically showclinical disease beginning ˜9 days. The AR1 protected against EAE asevidenced by reduced clinical disease (FIG. 10A).

In addition, C57BL/6 mice were nasally dosed with 50 μg myelinoligodendrocyte glycoprotein₂₉₋₁₄₆ genetically fused to pσ1 (MOG-pσ1) orto OVA-pσ1 (MOG:OVA-pσ1) three times at weekly intervals, and then oneweek after the last i.n. dose, mice were challenged s.c. with 150 μgMOG₃₅₋₃₃ on day 0 and 7 of challenge, and given i.v. PT on days 0 and 2.Both the MOG-pσ1 (n=5) and MOG; OVA-pσ1 (n=5) protected mice (p<0.001)when compared to PBS-dosed mice (n=5) (FIG. 10B).

Protection against PLP₁₃₉₋₁₅₁ challenge is attributed to the stimulationof the regulatory cytokines, IL-4, IL-10, and TGF-β. SJL mice were dosedwith AR1, OVA-pσ1, or PBS as described for FIG. 9. Mice were thenchallenged with PLP₁₃₉₋₁₅₁ peptide as described for FIG. 10. HNLN,spleens, and MLN were harvested at peak of disease (day 14) and purifiedCD4⁺ T cells were restimulated with PLP₁₃₉₋₄₅₁ peptide for two days, andevaluated in a cytokine ELISPOT. PBS- and OVA-pσ1-dosed (unprotectedmice) showed elevated (FIG. 11A) IFN-γ and (FIG. 11B) IL-17cytokine-forming cells (CFC), and no (FIG. 11C) IL-4, (FIG. 11D) IL-10,or (FIG. 11E) TGF-β CFC. In contrast, AR1-dosed (tolerized) mice showedelevated IL-4, IL-10, and TGF-β CFC and no IFN-γ or IL-17 CFC. Thus,only AR1 mice were protected against challenge, and tolerance induced toirrelevant protein (OVA-pσ1) did confer protection.

It was also determined that single nasal or oral dose with MOG-pσ1protects C57BL/6 mice against challenge with MOG₃₅₋₅₅. Mice (5/group)were dosed once (FIG. 12A) nasally or (FIG. 12B) orally with 10, 50, or100 μg of MOG₂₉₋₁₄₆-pσ1 (MOG-pσ1) or with PBS, and 10 days laterchallenged with MOG₃₅₋₅₅ per description for FIG. 10. In adose-dependent fashion, protection against autoimmune challenge showedprotection, but the 50 μg dose conferred the best protection with nodisease, while minimal disease was observed at the 10 or 100 μg doses.Thus, pσ1 delivery in the form of a fusion protein is an effective meansto deliver auto-antigens to the mucosa for the development of toleranceto self antigens.

To test whether pα1-mediated treatment could be therapeutic, a study wasperformed using MOG-pσ1 to stop further development of EAE. Four groups(5/group) of mice were induced with EAE as described in FIG. 10B. Then,groups were treated with MOG-pσ1 or PBS on day 7 or groups were treatedwith MOG-pσ1 or PBS on day 10 and 17. Results are shown in FIG. 13.Treatment with MOG-pσ1 demonstrated that protection against furtherdisease development can be conferred suggesting that pσ1-deliveredtoleragens can treat autoimmune diseases.

Significance Statement

Studies to date have mostly relied upon oral exposure to inducetolerance (Fujihashi et al., Acta. Odontol. Scand. 59:301-308, 2001;Mowat, Nature Rev. Immunol. 3:331-341, 2003; Weiner, Microbes &Infection 3:947-954, 2001) whereas most recently, studies have addressedthe potential of adapting i.n. delivery (Collins et al., Infect. Immun.70:2282-2287, 2002; Monfardini et al., J. Neuroimmunol. 123:123-134,2002; Winkler et al., Clin. Exp. Allergy. 32:30-36, 2002). The i.n.route clearly has a number of advantages, including less antigen doserequired, not subjecting the toleragen to alteration by the GI tract,and ease of delivery. In addition to considering route of delivery,efficient targeting of toleragens to mucosal inductive tissues couldreduce the amount of material needed for stimulation of toleranceregardless the route of delivery.

A particular strength of the system described herein is that it can beapplied to any number of toleragens that could be successfully fused topσ1, or another mucosal binding molecule as provided herein. Withoutmeaning to be limited to a single explanation, we propose that pσ1 cancircumvent the mucosal barrier and promote the uptake of toleragens bythe mucosal immune system, whether mediated via M cells, host epithelialcells, or their combination. Ultimately, T cell responsiveness willoccur in the mucosal inductive sites or draining mucosal LN. Thistoleragen delivery platform has promise in that a single oraladministration, and in some instances a single nasal application, canelicit tolerance.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. We thereforeclaim as our invention all that comes within the scope and spirit of thedescription, embodiments of which are described specifically in thefollowing claims.

1. A method of treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of a tolerizing fusion protein, wherein the tolerizing fusion protein comprises (1) a targeting portion comprising a reovirus protein σ1 (pσ1) and (2) an antigen to which tolerance is desired, thereby treating the autoimmune or inflammatory disease in the subject.
 2. The method of claim 1, wherein the autoimmune or inflammatory disease is multiple sclerosis, arthritis, diabetes, Hashimoto's disease, Graves' disease or Sjögren syndrome.
 3. The method of claim 2, wherein the autoimmune disease is multiple sclerosis.
 4. The method of claim 1, wherein the antigen comprises a protein, nucleic acid, carbohydrate, lipid or any combination thereof.
 5. The method of claim 4, wherein the antigen comprises a protein.
 6. The method of claim 5, wherein the antigen comprises a myelin protein and the autoimmune disease is multiple sclerosis.
 7. The method of claim 6, wherein the myelin protein is myelin oligodendrocyte glycoprotein (MOG).
 8. The method of claim 6, wherein the myelin protein comprises MOG₂₉₋₁₄₆.
 9. The method of claim 1, wherein the tolerizing fusion protein is administered in a single dose.
 10. The method of claim 9, wherein the single dose of the tolerizing fusion protein comprises 1 mg or less of the antigen.
 11. The method of claim 9, wherein the single dose of the tolerizing fusion protein comprises 500 μg or less of the antigen.
 12. The method of claim 9, wherein the single dose of the tolerizing fusion protein comprises 250 μg or less of the antigen.
 13. The method of claim 9, wherein the single dose of the tolerizing fusion protein comprises 100 μg or less of the antigen.
 14. The method of claim 1, wherein the tolerizing fusion protein is administered orally or nasally. 